ScFv Purification on Ni-column

Stixxer gcr at liv.ac.uk
Mon Dec 8 06:42:57 EST 1997


Dear all,

I recently was able to express a ScFv in a baculovirus system, however
in trying to purify the construct I am still unsuccesful. The ScFv has a
6x Histidine tag and the idea is to use it to purify on a Ni charged NTA
column. However when I put my cleared lysate on top of the column most
proteins including the ScFv run through without binding. My lysis buffer
is 10mM Tris-HCl pH 7.4, 0.1% Triton X-100, 10 mM Nacl. The buffers run
on the column are similar except for the Triton x-100. I've tried
raising the salt concentration (150 mM, 500 mM and even 1.5M) but to no
avail. Even going to a phosphate based lysis buffer (and hence
purification) did not help. Has anybody have some ideas on how to
overcome this problem, note that I do not like to denature my ScFv since
the whole reason for using the baculovirus system over E. coli was to
overcome folding problems. 

Allready thanks a lot ...
 
 
---------------------------------------------\/-----
Gert Roosen                                  /\ 
PhD Student                                 /--\
Department of Haematology                  (====\
Royal Liverpool University Hospital         \----)
Duncan Building 2nd floor                    \--/
Prescot Street L69 3BX Liverpool              \/
+ 44 (0)151 706 4326 (phone)                  /\ 
+ 44 (0)151 706 4311 (fax)                   /--\  
g.roosen at liv.ac.uk   (email)                /====)
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